Cancer development is greatly accelerated when DNA is not properly repaired during replication. Naturally occurring mutations in patients with Fanconi anemia have delineated a DNA repair pathway that is responsible for assuring genomic stability and preventing cancer formation. Fanconi anemia, a recessive genetic disease is characterized by a very high predisposition to developing cancer including acute myelogenous anemia, head and neck cancers, and gynecologic cancers. In addition, monoallelic mutations in the Fanconi anemia genes BRCA2 (FANCD1), PALB2 (FANCN), BRIP1 (FANCJ), and RAD51C (FANCO), predispose to breast and ovarian cancer. Cells derived from Fanconi anemia patients lack the ability to repair a very toxic type of DNA damage, interstrand crosslink, which covalently links the Watson and Crick strands of DNA together precluding their separation. This type of DNA damage may be caused by toxins in the environment, cellular processes metabolites or by chemotherapy during cancer treatment. Using a whole exome sequencing of a patient in the International Fanconi Anemia Registry, we have identified a mutation in a key cellular recombinase, RAD51. The patient was born with developmental abnormalities including an absent thumb. Bone marrow function is normal to date. Patient fibroblasts are hypersensitive to crosslinking agents but the chromosomal breakage levels following crosslinking agent exposure are only mildly elevated compared to the much higher levels seen in other Fanconi anemia patient fibroblasts. Although RAD51 foci formation is reduced and delayed following treatment with a range of DNA damaging agents, the patient fibroblasts do not exhibit sensitivity to ionizing radiation. In addition, the patient fibroblasts show wild type levels of sister chromatid exchanges following treatment with a DNA crosslinking drug and are capable of performing homologous recombination for repairing dysfunctional GFP gene. Collectively, these results suggest homology dependent repair of double strand breaks is not impaired in the patient cells. Additional experiments suggest that the essential function of RAD51 at the sites of stalled replication forks and quite possibly the key function necessary for prevention of tumorigenesis is to protect the DNA from being degraded, precluding correct repair. In the proposed experiments, we strive to understand the mechanism of function of RAD51 and its binding partners during crosslink repair. First, we propose to use the patient cell lines to understand homologous recombination-independent function of RAD51 and BRCA2 during DNA interstrand crosslink repair. Secondly, we want to understand the interplay between RAD51 and the Fanconi anemia pathway. Finally, we will study how the RAD51-interacting proteins affect the RAD51 function at the stalled replication fork. The overarching goal is to understand how the RAD51 and the associated factors might be preventing tumorigenesis by functioning during unperturbed DNA replication.